| Description | Inquire | The bacterial viability / toxicity detection kit contains two fluorescent dyes. Nucgreen is a green nucleic acid dye that can stain live and dead bacteria; Ethd III is a red nucleic acid dye that only stains dead bacteria with damaged cell membranes. When nucgreen and ethd III are properly mixed, The bacterial viability / toxicity detection kit contains two fluorescent dyes. Nucgreen is a green nucleic acid dye that can stain live and dead bacteria; Ethd III is a red nucleic acid dye that only stains dead bacteria with damaged cell membranes. When nucgreen and ethd III are properly mixed, the bacteria with intact cell membrane appear green, while the bacteria with damaged cell membrane can appear green and red under different channels, respectively. A common criterion for bacterial viability is the ability to propagate in a suitable nutrient medium, known as a growth assay. This kit is generally in good agreement with the growth assay results in liquid or solid medium. However, under certain conditions, membrane damaged bacteria may recover and propagate in nutrient medium, and such bacteria will be identified as dead bacteria in this assay. On the contrary, some bacteria with intact membranes may not be able to propagate in nutrient medium, but will be recognized as viable bacteria in this assay. Therefore, if there is a large difference between the test results of this kit and the bacterial growth assay, the above possibilities should be considered. Component： Product parameters： NucGreen: Ex/Em = 503/530 nm (结合 DNA)；EthD-III: Ex/Em = 530/620 nm (结合 DNA)。Usage:1 Preparation of control samples for live and dead bacteria (optional)1. Cultivate 4 mL of bacteria in liquid medium until late logarithmic phase.2. Prepare two 1 mL bacterial solutions in an EP tube and centrifuge for 10-15 minutes under 5000-10000 g conditions.3. Remove the supernatant and add 0.3 mL of 0.85% NaCl resuspended bacteria to one of the EP tubes, and 1 mL of 0.85% NaCl resuspended bacteria to the other tube.4. Add 0.7 mL of isopropanol to a tube containing 0.3 mL of 0.85% NaCl, and mix thoroughly (with a final concentration of 70% isopropanol) to prepare a dead bacterial sample.5. Incubate the two samples at room temperature for 1 hour and mix every 15 minutes.6. Centrifuge the two samples at 5000-10000 g for 10-15 minutes.7. Remove the supernatant, add 1 mL of 0.85% NaCl to resuspend the bacteria in both samples, and centrifuge again as in step 6.8. Use a spectrophotometer to measure the absorbance values (OD670) of two bacterial suspensions at 670 nm.9. Adjust the density of the two bacterial suspensions (live and dead) to 108 bacteria/mL (OD670 ≈ 0.3), and then dilute with 0.85% NaCl at 1:100 to achieve a final density of 106 bacteria/mL.10. Mix two bacterial suspensions as shown in the table below to obtain the required live cell ratio: dead cell ratio.Table 1 Mix live and dead bacterial suspensions by a certain volume to achieve the required ratio of live and dead cellsLive cells: Dead cellsVolume of viable bacterial suspension（mL）Volume of dead bacterial suspension（mL）0:10001.010:900.10.920:800.20.830:700.30.750:500.50.5100:01.00II Staining methods for fluorescence microscopy observation1. Mix 1 volume of component A, NucGreen, and 2 volumes of component B, EthD-III, in a microcentrifuge tube. After thorough mixing, add 8 volumes of 0.85% NaCl solution to obtain a 100 x dye solution.2. Every 100 µ L bacterial suspension, add 1 µ 100 x dye solution of L.3. Mix thoroughly and incubate at room temperature in the dark for 15 minutes.4. Take 5 µ The bacterial suspension after L staining was dropped onto a glass slide with an 18 mm square cover glass.5. Observe under a fluorescence microscope. The fluorescence of live and dead bacteria can be observed simultaneously under any standard FITC long-acting filter. Alternatively, live (green fluorescent) and dead (red fluorescent) bacteria can be observed using FITC and Cy3 (or Texas Red) channels, respectively.Attention: (1) Before staining bacteria, attention must be paid to removing residues of growth media. Nucleic acid and other media components can bind to NucGreen and EthD-III dyes in some way, resulting in unacceptable staining changes. A simple washing step is usually sufficient to remove interfering media components from bacterial suspension. It is not recommended to use phosphate buffer solutions as they can reduce staining efficiency. (2) Before starting the formal experiment, the dye concentration should be adjusted to distinguish between NucGreen labeling live bacteria and EthD-III labeling dead bacteria. The optimal concentration may vary depending on the bacterial strain. It is generally best to use the lowest dye concentration that can provide sufficient signal. The above conditions have been optimized for staining live/dead cells of Escherichia coli.III Before starting the staining method experiment of flow cytometry, please read the precautions under the fluorescence microscope staining steps.According to Table 1, add 11 different proportions of live and dead bacteria to the EP tube. Each of the 11 samples has a volume of 1 mL.2. Add 12 µ The A component of L, NucGreen, and 24 µ The B component EthD-III of L was mixed in a microcentrifuge tube. Add 3 to each of the 11 samples µ Mix the mixed dyes of L thoroughly by blowing them up and down several times. (Note: Additional control bacterial samples need to be prepared for separate NucGreen and EthD-III staining)3. Incubate at room temperature in the dark for 15 minutes.4. Analyze each sample using a flow cytometer, detect NucGreen positive cells using FITC channels, and detect EthD-III positive cells using PI or PE channels.Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. if the orifice plate is used for detection, a small amount of bacterial liquid can be left for imaging after standing for 10 min, which can effectively reduce the background. 3. in order to be closer to the real results, it is recommended to keep the brightness of red fluorescence consistent with that of green fluorescence in merge pictures. 4. fluorescent dyes have quenching problems. Please try to avoid light during experimental operation to slow down fluorescence quenching. 5. for your safety and health, please wear experimental clothes and disposable gloves.Scope of application：Staining of dead and live bacteria... Read More | Product content: S665546Component50 TStorageS665546ABuffer QSL45 mLRTS665546BBuffer RIL11 mL2-8℃S665546CBuffer ML10 mLRTS665546DBuffer GW1 (concentrate)13 mLRTS665546EBuffer GW2 (concentrate)26 mLRTS665546FBuffer EBL13 mLRTS665546GRNase A240 µLRTS665546HLysis Tubes Ⅱ50 Product content: S665546Component50 TStorageS665546ABuffer QSL45 mLRTS665546BBuffer RIL11 mL2-8℃S665546CBuffer ML10 mLRTS665546DBuffer GW1 (concentrate)13 mLRTS665546EBuffer GW2 (concentrate)26 mLRTS665546FBuffer EBL13 mLRTS665546GRNase A240 µLRTS665546HLysis Tubes Ⅱ50 EARTS665546ISpin Columns DM With Collection Tubes50 EARTProduct IntroductionThis kit provides a method for extracting total DNA from soil or fecal samples, including the total DNA of cells, bacteria, parasites, and viruses in the samples. It is also suitable for extracting DNA from samples containing high concentrations of PCR reaction inhibitors. This reagent kit adopts a unique buffering system to efficiently bind DNA from the lysis solution to the adsorption column. Inhibitors of PCR and enzyme reactions, as well as residual impurities, can be effectively removed through washing steps. Finally, high-purity DNA can be obtained by washing with low salt buffer or water. The purified DNA can be directly used for downstream experiments such as second-generation sequencing (16S amplicons and metagenomes), library construction, PCR, qPCR, Southern Blot, enzyme digestion molecular markers, etc.Self prepared reagents1. Constant temperature mixer - Product number: CW25932. Anhydrous ethanol, isopropanol3. Vortex oscillator or tissue grinderPreparation and important precautions before the experiment1. Samples should avoid repeated freeze-thaw cycles, otherwise it may result in smaller extracted DNA fragments and a decrease in extraction volume.2.Before the first use, anhydrous ethanol should be added to Buffer GW1 (concentrate) and Buffer GW2 (concentrate) according to the instructions on the reagent bottle label.3. Take out the buffer RIL before use and store it at 2-8 ℃ immediately after use.Operation steps1. Centrifuge the Lysis Tube briefly to allow the beads to settle at the bottom.2. a. Add 0.1-0.3 g of soil or fecal sample to Lysis Tube, and add 740-820 µ L Buffer QSL and 4 µ L RNase A, tighten the tube cover and briefly vortex to mix.b. If fecal samples are stored in non lytic fecal preservation solutions (such as CWY041S and CWY041M), add 200 to Lysis Tube µ L-600 µ L solid-liquid mixture, centrifuge at 13000 rpm for 1 minute, discard the storage solution (if the amount of solid after centrifugation is too small, it can be enriched again, but should not exceed 0.3g). Join 620 µ LBuffer QSL and 4 µ L RNase A, tighten the tube cover and briefly vortex to mix.3. Fix the Lysis Tube in an oscillating grinding device equipped with a 2 mL adapter and process it according to the optimized grinding conditions of your equipment (see appendix).4. Shake the Lysis Tube on a constant temperature mixer at 70 ℃ and 1200 rpm for 10 minutes. Subsequently, centrifuge at 13000 rpm for 2 minutes to precipitate solid particles. Transfer 540 µ Transfer the supernatant to a new 2 mL centrifuge tube.5. Add 180 µ L Buffer RIL, vortex for 5 seconds, centrifuge at 13000 rpm for 2 minutes.Attention: Remove the buffer RIL before use and store it at 2-8 ℃ immediately after use.6. Add 160 to the new centrifuge tube in sequence µ L Buffer ML, 480 µ Supernatant from step 5, 320 µ L isopropanol, vortex for 5 seconds.7. Transfer the solution from the previous step to 650 µ Centrifuge at 12000 rpm (~13400 × g) for 1 minute into the spin columns DM that have been loaded into the collection tube.8. Discard the waste liquid in the collection pipe and place the adsorption column back into the collection pipe. Repeat step 7 until all the solution has been transferred.9. Add 500 to the adsorption column µ L Buffer GW1 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.10. Add 500 to the adsorption column µ L Buffer GW2 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube. 11. Repeat step 10.12.12000 rpm for 2 minutes and discard the waste liquid from the collection tube. Place the adsorption column at room temperature for a few minutes to thoroughly air dry.Note: The purpose of this step is to remove residual ethanol from the adsorption column, which can affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).13. Place the adsorption column in a new centrifuge tube (self provided) and add 50-200 drops of suspended droplets to the middle of the adsorption column µ L Buffer EBL or sterilized water, leave at room temperature for 2-5 minutes, centrifuge at 12000 rpm for 1 minute, collect DNA solution, and store DNA at -20 ℃.Note: 1) Incubating at room temperature for 5 minutes before centrifugation can increase yield.2) Use an additional 50-100 µ Further elution with L buffer or sterilized water can increase yield.3) If you want to increase the final concentration of DNA, you can add the DNA eluent obtained in step 13 back onto the adsorption membrane and repeat step 13, but it may reduce the total yield.4) The elution buffer does not contain chelating agents, please store DNA at -20 ℃.5) The residual trace PCR inhibitors in the genomic DNA template may have adverse effects on the PCR reaction, which can usually be resolved by diluting the DNA by 2-10 times.Appendix: Grind the sample using one of the following methods1. Manually vortex oscillate at maximum speed on the vortex oscillator for 10 minutes.2. On a vortex oscillator equipped with a 1.5-2 mL horizontal centrifuge tube holder, oscillate at maximum speed for 10 minutes (keeping the Lysis Tube horizontal). If the sample size exceeds 12, extend by 5-10 minutes. For example, using Scientific Industries or Mobile's Vortex Genie2 vortex oscillator.3.When using Qiagen's TissueLyser II, grind at 25Hz for 10 minutes.4.When using Qiagen's PowerLyzer 24 Homogenizer, homogenize at 2000 rpm for 30 seconds, pause for 30 seconds, and then homogenize again at 2000 rpm for 30 seconds.5.When using FastPrep-24 from MP Biomedicals, the recommended speed is 6.0 and the time is 40 seconds... Read More | The Succinic Acid (Succinate) assay kit is suitable for the specific assay of succinic acid in wine, cheese, eggs, sauce and other food products. Succinic acid (or succinate) is found in all plant and animal materials as a result of the central metabolic role played by this dicarboxylic acid in the The Succinic Acid (Succinate) assay kit is suitable for the specific assay of succinic acid in wine, cheese, eggs, sauce and other food products. Succinic acid (or succinate) is found in all plant and animal materials as a result of the central metabolic role played by this dicarboxylic acid in the Citric Acid Cycle. Succinic acid concentrations are monitored in the manufacture of numerous foodstuffs and beverages, including wine, soy sauce, soy bean flour, fruit juice and dairy products (e.g. cheese).Product Description： Succinic acid is found in all plant and animal materials as a result of the central metabolic role played by this dicarboxylic acid in the Citric Acid Cycle. Succinic acid concentrations are monitored in the manufacture of numerous foodstuffs and beverages, including wine, soy sauce, soy bean flour, fruit juice and dairy products (e.g. cheese). The ripening process of apples can be followed by monitoring the falling levels of succinic acid. The occurrence of > 5 mg/kg of this acid in egg and egg products is indicative of microbial contamination. Apart from use as a flavouring agent in the food and beverage industries, succinic acid finds many other non-food applications, such as in the production of dyes, drugs, perfumes, lacquers, photographic chemicals and coolants. Preparation Instructions：Suitable for succinate determination in food, beverage, agricultural products, and other biological samples.Note for Content：The number of manual tests per kit can be doubled if all volumes are halved. This can be readily accommodated using the MegaQuantTM Wave Spectrophotometer (D-MQWAVE).Browse all of our organic acid assay kits.Principle：The Succinate Assay Kit provides a simple, one step assay for measuring succinate. In this assay succinate is converted to pyruvate which reacts with specific reagents and dye to form a colored product. The color intensity at 570 nm or fluorescencAdvantages：Extended cofactors stability. Dissolved cofactors stable for > 1 year at 4oC.Very competitive price (cost per test)All reagents stable for > 2 years as suppliedVery rapid reaction (even at room temperature)Mega-Calc™ software tool is available from our website for hassle-free raw data processingStandard includedSuitable for manual, microplate and auto-analyser formats... Read More | The content of this cell is too long for an XLSX file (more than 32767 characters). Please use the CSV format for this export |